SUPPLEMENTAL MATERIAL

Pharmacokinetics, Biotransformation, and Excretion of

[14C]Etelcalcetide (AMG 416) Following a Single

Microtracer Intravenous Dose in Patients With Chronic

Kidney Disease On Hemodialysis

Raju Subramanian, Xiaochun Zhu, M. Benjamin Hock, Bethlyn J. Sloey, Benjamin Wu,

Sarah F. Wilson, Ogo Egbuna, J. Greg Slatter, Jim Xiao, Gary L. Skiles

Amgen Inc., Thousand Oaks, California, USA

Address correspondence to: Raju Subramanian

Amgen Inc.

One Amgen Center Drive

Thousand Oaks, CA 91320

Phone: 805-447-6301

Fax: 805-447-1010

Email: rajus@amgen.com

Target journal: Clinical Pharmacokinetics

Subramanian et al 1

SUPPLEMENTAL METHODS

Sample Collection and Processing

Blood samples were collected into potassium-EDTA for [14C]etelcalcetide-derived

radioactivity (referred to as the “total C-14”) analysis, etelcalcetide and total M11 (TM11)

bioanalysis, and biotransformation product profiling according to the schedule described.

An aliquot of whole blood from each time-point was saved for carbon-14 (C-14)

analysis. The remainder was processed to plasma by centrifuging the whole blood at

1500×g for 15 minutes at 4°C. An aliquot of plasma was set aside for C-14 analysis. The

remaining plasma was acidified with citric acid (20mg/mL) to prepare acidified aliquots

for bioanalysis and biotransformation product profiling. The prepared non-acidified and

acidified plasma was stored at −70°C.

The discharged urine from each patient was collected in 24-hour intervals during the in-

clinic period (pre- and postdose; study days 1 to 11). The weight of the total urine in

each 24-hour period was recorded and aliquots of this pooled urine were stored at

−70°C for further C-14 analysis and biotransformation product profiling.

Total dialysate was collected for C-14 analysis and radioprofiling at each hemodialysis

session for (1) for each patient during the in-clinic period (study days 1 to 11) and

outpatient days (study days 14 to 39), and (2) for select patients (n=4) at the six time-

points in the extended pharmacokinetic collection period. The dialysate from each

hemodialysis session was collected in collection barrels in approximately 1-hour

intervals and their weight was recorded for each interval. After thorough mixing, a fixed

percentage of aliquots from each interval were combined and aliquots of this pooled

dialysate were stored at −70°C for C-14 analysis and biotransformation product profiling.

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The feces from each patient was collected during the in-clinic period (pre- and postdose;

study days 1 to 11). The total weight of each discharge was recorded at the clinic and

stored at −70°C until shipment to Covance (Madison, WI). At Covance, the feces

samples were combined by patient at 24-hour intervals, combined with appropriate

volume of ethanol/water (1:1 v/v) mixture and homogenized. A sub-sample of each

homogenized sample was freeze-dried, and then ground in a pestle and mortar; these

were used for C-14 analysis. The freeze-dried samples were stored at room

temperature. Aliquots of homogenized feces were stored at −70°C for biotransformation

product profiling.

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Bioanalysis of Etelcalcetide and Total M11

Plasma concentrations of etelcalcetide and TM11 were determined by liquid

chromatography-tandem mass spectrometry (LC-MS/MS) method. Etelcalcetide

concentrations were measured in acidified plasma by an electrospray ionization (ESI)

positive-ion mode LC-MS/MS assay at Alturas Analytics (Moscow, ID). The validated

method used solid phase extraction; the MS response was linear over the range of

0.2─100 ng/mL (lower limit of quantification [LLOQ], 0.2 ng/mL). Stable isotope-labeled

etelcalcetide was used as the internal standard. Sample extracts were injected into a

high-performance liquid chromatography (HPLC)-MS/MS triple quadrupole mass

spectrometer (Sciex API5500 or 6500; Sciex, Farmingham, MA). A Discovery HS-C18,

3-µm HPLC column (2.1×50 mm; Supelco, Bellefonte, PA) was used to separate

etelcalcetide and the internal standard from interfering compounds. The peak area of

the product ion of etelcalcetide was measured against the peak area of the product ion

of the internal standard.

TM11 is the sulfhydryl (reduced) form of the D-amino acid peptide backbone in

etelcalcetide [5]. TM11 was liberated by chemical reduction of all etelcalcetide-related

mixed disulfides with an intact D-amino acid backbone present in plasma. Reduction

was performed with tris(2-carboxyethyl)phosphine (TCEP), a disulfide bond reducing

agent. TM11 concentrations were measured by ESI positive-ion mode LC-MS/MS at

Amgen Inc. Acidified plasma was reduced with TCEP followed by protein precipitation

with heptafluorobutyric acid in 50:50 acetonitrile:methanol. The MS response was linear

over the TM11 concentration range of 50─2000 ng/mL (LLOQ, 50 ng/mL); stable

isotope-labeled etelcalcetide was used as the internal standard. Sample extracts were

separated by reversed-phase LC using a gradient elution, followed by LC-MS/MS (Sciex

API 4000).

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Accelerator Mass Spectrometry

Sample Preparation

The C-14 content in whole blood, plasma, urine, and fecal samples was determined by

accelerator mass spectrometry (AMS). All samples underwent a graphitization

procedure before C-14 detection by AMS. Carbon carrier (sodium benzoate) was added

as necessary during sample preparation to achieve approximately 2 mg of carbon.

Urine samples were processed with a 10-fold dilution and a 100-µL aliquot volume;

select plasma samples that were too concentrated for direct AMS analysis were

processed with a 10-fold dilution; blood (20-µL aliquots) and lyophilized feces (3- to 4-mg

aliquots) were directly analyzed by AMS.

C-14 detection by AMS

The prepared cathodes were placed in the ion source of a single-stage AMS (National

Electrostatics Corp., Middleton, WI) using a Cs+ ion beam, and the 14C:12C ratio of all

sources of carbon in the sample was determined. The prepared quartz sample tubes

were heat-sealed under vacuum and then heated for 2 hours at 900°C to oxidize all

carbon in the sample to CO2. The CO2 was then cryogenically transferred and sealed

into an evacuated glass tube containing TiH2 and Zn (reducing agent), with cobalt

powder as catalyst. Samples were heated for 4 hours at 500°C, then for 6 hours at

550°C, reducing CO2 to solid carbon (graphite). The resultant graphite was pressed into

aluminum cathodes. When possible, background 14C:12C ratios were measured from

predose sample and subtracted from the ratios determined for postdose sample. The

lower limit of quantification for C-14 values obtained following AMS analysis was

dependent on the carbon content and dilution factor of the samples. The limits of

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quantification, in ng equivalents of etelcalcetide/(mL or g), were approximately as

follows: plasma, 0.65; whole blood, 1.32; vomit, 0.57; dialysate, 0.009; urine, 0.28; and

feces, 5.77.

Biotransformation Product Profiling

Sample Preparation

For each biomatrix, samples from all patients were pooled to obtain a single sample

representative of all patients in the study. For urine, the weights of individual aliquots

included in the pool (study day 1 to 10) were proportional to the weight of sample

collected in each study day from each patient. A grand pool representing all patients

was then prepared by combining the individual patient pool in proportion to the total

weight of urine, and then acidified with FA (final 1% v/v). For dialysate, the weights of

individual aliquots included in the pool were proportional to the weight of sample

collected in study day 4 from each patient and this pool was acidified with formic acid

(final 1% v/v). For plasma, volume aliquots of acidified plasma at each time-point were

pooled proportional to the time interval to approximate an AUC pool from 0 to start of

first dialysis session postdose (approximately 68 hours) and another AUC pool from end

of first dialysis session (approximately 72 hours) to the start of the second dialysis

session postdose (approximately 116 hours). Equal volumes of this AUC pool were then

combined to prepare one representative sample, one for AUC0-68hr and another for AUC72-

116hr, across all patients.

Urine: The pooled urine sample was concentrated by a solid phase extraction (SPE)

procedure. The SPE column (Oasis WCX 6cc, 150 mg, 30 μm, Waters Corp., Milford,

MA) was conditioned with methanol (3 mL) and 5 mM ammonium acetate (3 mL),

respectively. Pooled human urine (4809 μL) was then loaded onto the column. The

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column was then washed with 5 mM ammonium acetate (4 mL) and acetonitrile/water

(1:4 v/v; 4 mL) sequentially. The column content was finally eluted down with

methanol/trifluoroacetic acid (v/v=98/2; 8 mL), dried by nitrogen gas overnight at 23°C.

The residue was reconstituted into 300 μL of 0.1% formic acid and analyzed by liquid

chromatography–high-resolution mass spectrometry (LC-HRMS). Recovery of the

eluted radioactivity from the SPE was approximately 84%. For tris(2-carboxyethyl

phosphine (TCEP) reduction, pooled urine was first incubated with TCEP (final 10 mM)

at 37°C for 1.5 hours and then prepared by SPE as described above.

Plasma: Area under the plasma concentration-time curve (AUC) pooled plasma was

subjected to a protein precipitation procedure. The supernatants obtained following

protein precipitation were concentrated to enable LC-HRMS profiling. Pooled samples

(3 mL) were treated with 3 mL of acetonitrile/methanol (1:1 v/v) and vortex-mixed for

approximately 1 minute. The resulting suspension was centrifuged for 20 minutes at

1500×g. The supernatant was transferred to a 15-mL Falcon tube. The extraction

procedure was repeated once more, and the combined supernatants were dried down to

~300 μL in a vacuum centrifuge (SpeedVac; Savant SPD2010; Thermo Fisher Scientific,

Waltham, MA). The concentrated supernatant was analyzed by LC-HRMS. For TCEP

reduction, 1 mL of pooled plasma was first diluted with 2 mL of 0.1% aqueous

trifluoroacetic acid and then incubated with TCEP (final 10 mM) at 37°C for 1.5 hours.

The reduced plasma was processed the same as above.

For sample preparation of SAPC for LC-HRMS analysis, AUC pooled human plasma (1

mL) was centrifuged at 20,817×g for 5 minutes; the top layer was transferred to a new

centrifuge tube, diluted with 0.5 mL of 20 mM pH 7.1 sodium phosphate buffer (Buffer

A), and vortex-mixed. This diluted plasma was loaded onto a buffer-exchange column

(Econo-Pac 10 DG column; Bio-Rad, Hercules, CA). The column was then eluted five

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times (1 mL each time) with Buffer A. Five 1-mL fractions (labeled fractions 1–5) were

collected. Fractions 2 to 4 were loaded onto an albumin affinity gel column (Affi-Gel®

column, 153-7301; Bio-Rad). The affinity gel column was then washed with 25 mL of

Buffer A to remove nonalbumin proteins and small molecules. Subsequently, 20 mL of

NaSCN phosphate buffer (1.5 M NaSCN, 20 mM sodium phosphate in water, pH 7.1)

was used to elute the serum albumin protein. The eluting fractions (20 mL) were loaded

onto a Vivaspin 20 tube (molecular weight cutoff, 10,000 Da; Sartorius Stedim Biotech

GmbH; Goettingen, Germany) and centrifuged at 2250×g (20°C) to remove the salts.

After being washed with water (20 mL each time) three times, the remaining desalted

solution (~2.5 mL) was transferred to a 4-mL vial and lyophilized using a ModulyoD

Freeze Dryer (Thermo Fisher Scientific Inc.; San Jose, CA). An aliquot of dried human

serum albumin sample (0.33 mg) was dissolved into 495 μL of buffered solution (pH 5.5)

containing urea (4M), ammonium acetate (50 mM), and hydroxylamine (20 mM) and

digested via addition of 16.5 μL of Lys-C (1 μg/μL) with a substrate:enzyme ratio of 1:20

(w/w). The digestion solution was gently vortex-mixed and incubated at 37°C for 24

hours. The digestion reaction was stopped by addition of 5.1 μL of formic acid (1% v/v

final solution). The digest was then analyzed by LC-HRMS

Dialysate : The pooled dialysate sample (49.7 mL) was dried down to approximately 5

mL in a SpeedVac at 35°C. The concentrate was processed by the urine SPE method,

and the reconstituted solution (300 μL) was analyzed by LC-HRMS. Recovery of the

eluted radioactivity from the SPE was approximately 91%. For TCEP reduction, the

pooled dialysate (49.7 mL) was incubated with TCEP (final 10 mM) at 37°C for 1.5

hours. The TCEP-treated dialysate was processed the same as above.

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LC─Fraction Collection+AMS and LC–High-Resolution MS

Radioprofiles by LC─fraction collection followed by off-line AMS (LC-FC+AMS) and

biotransformation product identification by LC–high-resolution mass spectrometry

(HRMS) were performed on pooled samples of plasma, urine, and dialysate with and

without TCEP reduction at Xceleron and Amgen Inc., respectively. Samples from all

patients were pooled to obtain a representative sample, which was acidified with formic

acid (1% v/v). Formation of serum albumin peptide conjugate (SAPC) was confirmed by

LC-HRMS analysis of the lysyl endopeptidase (Lys-C; Wako Chemicals USA, Inc.,

Richmond, VA) digest of SAPC in pooled human plasma.

LC-HRMS analysis of plasma, urine, dialysate, and SAPC in plasma was performed as

previously described [13]. For C-14 biotransformation profiling by LC-FC+AMS, samples

were prepared as follows: pooled urine was centrifuged for 10 minutes at 3210×g at 4°C

and supernatants were used for LC-FC+AMS. Pooled plasma was diluted eight-fold with

aqueous formic acid (0.2%, v/v) and centrifuged for 10 minutes at 3210×g at 4°C; the top

layer was used for LC-FC+AMS; direct injection of diluted plasma was performed to

enable detection of protein conjugates. Pooled dialysate (5 mL) was dried to

approximately 0.5 mL in a vacuum centrifuge (SpeedVac) at ambient temperature, and

the concentrate was used for LC-FC+AMS. TCEP-reduced samples of urine, plasma,

and dialysate were generated as described for LC-HRMS.

LC-FC+AMS analyses used the same LC components and methods as described for

LC-HRMS, with smaller injection volumes (urine and dialysate, 80 µL; plasma, 20 µL);

the flow post column was set completely for fraction collection. Fifteen-second fractions

were collected from each LC run (single injection runs for urine and dialysate; five

injections for plasma, with fractions pooled from each run before AMS sample

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processing). Fractions were pooled based on LC-HRMS chromatograms to optimize

peak resolution and prepared for graphitization and C-14 determination by AMS.

LC-HRMS Analysis of Human Serum Albumin Digest by Lys-C

The LC-HRMS system was an Agilent 1200 (Agilent Technologies, Santa Clara, CA) LC

system with a binary pump, photodiode array detector, a degasser, a refrigerated

autosampler, and a column heater and in-line positive ionization electrospray ionization

(ESI) HRMS (LTQ Velos Orbitrap; Thermo Fisher, Waltham, MA). Data were acquired

using Xcalibur software (version 2.1).

Samples (50 µL) were injected onto a Vydac 218MS52 column (250×2.1 mm; Grace;

Deerfield, IL) maintained at 40°C and eluted with a gradient method that used mobile

phases A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) as follows:

0 to 2 minutes, 3% B, 0.2 mL/min; 2 to 45 minutes, 3% to 40% B, 0.2 mL/min; 45 to 46

minutes, 40% to 80% B, 0.2 to 0.3 mL/min; 46 to 58 minutes, 80% B, 0.3 mL/min; 58 to

60 minutes, 80% to 3% B, 0.3 to 0.2 mL/min; 60 to 65 minutes, 3% B, 0.2 mL/min.

MS analysis of the Lys-C digests was performed using positive ionization ESI HRMS

(LTQ Velos Orbitrap; Thermo Fischer Scientific, Inc.) in-line using full-scan mode (mass-

to-charge ratio [m/z] 200–1000) at a resolution of 30,000, followed by collision induced

dissociation MS/MS scan event with a resolution of 7500 at 35% collision energy.

MS/MS scan was implemented to either the most abundant ion observed in the full-scan

spectrum or predefined m/z list.

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Pharmacokinetic Analysis

Apparent terminal half-life (t½,z) was calculated as t½,z=ln(2)/λz, where λz was the first-

order terminal rate constant estimated via linear regression of the terminal log-linear

decay of the concentration-time profile from the time points acquired immediately before

the start of hemodialysis sessions.

The ratio of the total (TM11) AUC to the total C-14 AUC was calculated as

AUCTM11/AUC[14C]etelcalcetide; the ratio of etelcalcetide AUC to the total C-14 AUC was

calculated as AUCetelcalcetide/AUC[14C]etelcalcetide.

Nonhemodialysis pharmacokinetics parameters analyzed included maximum plasma

concentration (Cmax); time to Cmax (tmax); the last observed plasma concentration (Clast);

time to Clast (tlast); the area under the plasma concentration-time curve (AUC) from time 0

on study day 1 (predose) to start of hemodialysis on study day 4 (3 days postdose;

AUC3d) and from time 0 on study day 1 (predose) to study day 11 (10 days postdose;

AUC10d), estimated using the linear trapezoidal method; apparent terminal half-life (t½,z;

the ratio of TM11 AUC to the total C-14 AUC; and the ratio of etelcalcetide AUC to the

total C-14 AUC.

Additional pharmacokinetics parameters from the hemodialysis period based on

individual plasma etelcalcetide pharmacokinetics samples included hemodialysis

clearance (CLHD) and the hemodialysis extraction ratio (EHD) using the following formulas:

CLHD = EHD × QP

EHD = (AUCA,HD – AUCV,HD)/AUCA,HD

where AUCA,HD is the area under the arterial plasma concentration-time curve

obtained during hemodialysis session on study day 4; AUCV,HD is the area under the

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venous plasma concentration-time curve obtained during hemodialysis session on

study day 4; and QP is the plasma flow rate through the dialyzer, calculated using the

following equation:

QP = QB × (1 − HCT)

where QB is the blood flow rate through the dialyzer during hemodialysis on study

day 4 and HCT is the hematocrit count obtained on study day −1.

Anti-Etelcalcetide Antibody Analysis

A validated dual-flow cell immunoassay was used to screen for and confirm the

presence of anti-etelcalcetide and anti-SAPC antibodies. For both screening and

confirmatory assays, etelcalcetide and SAPC were separately immobilized onto

carboxymethyl-dextran coated sensor chip flow cell surfaces in a Biacore™ 3000

instrument (GE Healthcare Bio-Sciences, Pittsburgh, PA). In the screening assay, serum

specimens were injected over both surfaces to initially detect etelcalcetide-reactive

antibodies. Confirmation that the sample binding was due to etelcalcetide-reactive

antibodies was demonstrated by injecting a solution of goat anti-human (IgA+IgG+IgM)

antibody after each human specimen injection. A specimen was considered positive if

both the screening and confirmatory reactivity was greater than the assay cut point on

either surface. Sensitivities for anti-etelcalcetide antibody detection on etelcalcetide and

SAPC surfaces were 1289 and 244 ng/mL, with lower limits of reliable detection of 2000

and 500 ng/mL, respectively.

Subramanian et al 12

SUPPLEMENTAL TABLE

Supplemental Table 1. Summary of Cumulative Total C-14 Excretion Ratios in Urine and Feces on Nonsampled Days

Patient

1 2 3 4 5 6

Cumulative Excretion (% of Administered C-14 Dose)

CED, SD4 to SD11a 17.63 16.49 11.66 10.27 14.61 16.55

CED, SD14 to SD176a 44.11 47.75 38.66 NCb NCb 45.35

CEU, SD1 to SD10a 0.34 0.18 2.44 4.26 3.47 0.07

CEF, SD1 to SD10a 1.07 1.07 1.19 0.29 1.10 1.34

Ratio of Cumulative Excretion (Study Days 1 to 11)

CEU, SD1 to SD10/CED, SD4 to SD11 0.02 0.01 0.21 0.42 0.24 0.00

Subramanian et al 13

Patient

1 2 3 4 5 6

CEF, SD1 to SD10/CED, SD4 to SD11 0.06 0.06 0.10 0.03 0.08 0.08

CE=cumulative excretion in respective matrices (U=urine; F=feces; D=dialysate); SD=study day.

aSee Materials and Methods section 2.8.

bExcretion during extended pharmacokinetics collection period not collected; hence, value not computed.

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SUPPLEMENTAL FIGURE LEGENDS

Supplemental Fig. 1 Radioactivity excretion rate in dialysate following

administration of a single intravenous dose of

[14C]etelcalcetide (10 mg; 26.3 kBq) to patients with CKD on

hemodialysis (n=6). CKD=chronic kidney disease

Supplemental Fig. 2 HPLC radiochromatogram of pooled urine (0–240 hours)

following a single intravenous dose of [14C]etelcalcetide (10

mg; 26.3 kBq) to patients with CKD on hemodialysis.

CKD=chronic kidney disease; dpm=disintegrations per minute;

HPLC=high-performance liquid chromatography

Supplemental Fig. 3 HPLC radiochromatogram of TCEP treated plasma, dialysate

and urine obtained following a single intravenous dose of

[14C]etelcalcetide (10 mg; 26.3 kBq) to patients with CKD on

hemodialysis. (a) AUC pooled plasma (0–68 hours), (b) pooled

dialysate from the first hemodialysis session, (c) pooled urine

(0–240 hours). CKD=chronic kidney disease; HPLC=high-

performance liquid chromatography; TCEP=tris(2-carboxyethyl

phosphine); TM11=total M11

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Supplemental Fig. 1

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Supplemental Fig. 2

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Supplemental Fig. 3

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